The invention relates to a method and an apparatus for determining fluorophores on objects, particularly on the living ocular fundus.
It is known in the art to use the natural fluorescence of objects to analyze these objects, for example to evaluate the metabolic state of living biological tissue, such as the ocular fundus in ophthalmological examinations. Exciting fluorescence in these objects thus makes it possible by optical evaluation of the autofluorescence in biological tissue to draw conclusions regarding the metabolic state of this tissue in a non-invasive manner. This requires an at least two-dimensional representation of the distribution of different fluorophores in the examined biological object. The examination of the vascular system of the ocular fundus in ophthalmological examinations by means of fluorescence markers, e.g., using intravenously injected sodium fluorescein (quantum efficiency approximately 1) or indocyanine green is state of the art in clinical routine. Autofluorescence on the ocular fundus can be measured only with very low quantum efficiency, with minor concentrations of fluorophores being present in addition. An added difficulty is that the limits for maximum permissible light exposure (American National Standard for Use of Lasers, ANSI 136.1xe2x80x941993) must be complied with in the excitation of autofluorescence. U.S. Pat. No. 4,569,345 proposes to evaluate oxygenation of the retina by measuring autofluorescence at 520 nm and 540 nm, with excitation occurring at 450 nm. The disadvantage is that excitation at 450 nm results in absorption in the lens causing the lens to be excited to fluorescence. Based on the fluorescence measurements at 520 nm and 540 nm, it should be possible to compensate the influence of the ocular media, such as the lens, the aqueous humor, the cornea, and the vitreous body. Since the fluorescence of the lens is more intense than the fluorescence of the ocular fundus by approximately three orders of magnitude, it is almost impossible to obtain analyzable fluorescence signals from the ocular fundus in narrow wavelength ranges and to use these to compensate the influence of the ocular media. Radiation at 450 nm also excites lipid peroxidation products to fluorescence, which also show a measurable fluorescence at 540 nm. This means that the influences of lens fluorescence, of flavoproteins of the ocular fundus and of lipid peroxidation products on the ocular fundus cannot be determined separately by measuring the fluorescence at two wavelengths even if the fluorescence spectra of the individual fluorophores are known.
U.S. Pat. No. 4,213,678 discloses the principle of confocal scanning of the ocular fundus with a laser scanning ophthalmoscope. According to this principle, reflected images of the ocular fundus are obtained after a continuously radiant laser beam scanningly illuminates the ocular fundus and the reflected light is recorded by a detector. This technique, using fluorescent markers (sodium fluorescein, ICG), makes it possible to evaluate the retinal or choroidal vascular system. Furthermore, v. Rxc3xcckmann et al. (Br. J. Ophthalmol, 1995; 79:407-412) have shown that particularly in age-related macular degeneration (AMD), autofluorescence can be detected if the ocular fundus is excited with 488 nm wavelength light and the total fluorescence light, globally above a 520 nm cut-off wavelength, is integrally recorded. Superimposing several autofluorescence images improves the signal-to-noise ratio.
Various procedures for characterizing primarily the pathological state by means of the autofluorescence spectra of specific fluorophores, e.g., in age-related macular degeneration (AMD), have been published. By exciting a relatively large area on the ocular fundus and measuring the autofluorescence spectrum, Delori et al. (Invest Ophthalmol Vis Sci., 1995; 36:718-729) were able to show that the degradation product of lipid peroxidation, lipofuscin, largely determines the autofluorescence in AMD.
Using imaging spectrometry in which the autofluorescence spectra of all excited sites of the ocular fundus are measured simultaneously along a line after excitation to autofluorescence, Schweitzer et al. (Invest. Ophthalmol Vis Sci., 1998; 38:387) showed that after excitation in various wavelength ranges autofluorescence can be excited for at least two fluorophores on the ocular fundus. While shorter wave autofluorescence is more pronounced in healthy eyes, a more pronounced long wave fluorescence spectrum is evident in AMD. Biological tissue, particularly that of the ocular fundus, has the characteristic that any possible radiation within the visible spectral region excites several fluorophores, the fluorescence spectra of which overlap. The quantum efficiency of the natural fluorophores is thereby very low. Since the ocular fundus contains the light-sensitive receptors of the eye, the excitation intensity must be small enough so as to exclude any damage. A clinical application of autofluorescence to characterize the metabolic state or its change at a given instant before morphological changes are detected furthermore requires a two-dimensional autofluorescence measurement with the highest possible spatial resolution, which also causes a reduction in the fluorescence light that can be detected from each site. The finer the spatial resolution per measuring point, the lower the measurable fluorescence intensity will be. An added problem particularly for measuring autofluorescence on the ocular fundus is the fact that the eye is a movable object that is able to focus on an object for only a limited time. The consequence of these particular examination conditions is that it is currently practically impossible to separate the fluorophores active on the ocular fundus by the different fluorescence spectra due to the overlapping fluorescence spectra and the poor signal-to-noise ratio with which autofluorescence on the ocular fundus can be measured.
There is considerable interest among persons skilled in the art, however, in the two-dimensional measurement of the autofluorescence of different fluorophores, especially on the ocular fundus, to identify the metabolic state, which is effectively impossible with the known methods and under the aforementioned special ophthalmological conditions.
It is therefore the aim of the invention to distinguish reliably at least partially overlapping fluorophores of objects in excitation and/or fluorescence spectra, even if fluorescence intensities are very low and to select them for analysis with the possibility of a two-dimensional representation.
In accordance with the invention, a method and an apparatus are proposed, with which the object, for example, the ocular fundus for ophthalmological examinations, is subjected to point-to-point illumination by pulsed laser light and excited to autofluorescence with two-dimensional extension. The transient fluorescence light created after excitation by each laser pulse is detected in time-correlated single photon counting. Based on the time fluorescence behavior for each site determined by time-correlated single photon counting, the fluorescence time constants are calculated. Since the fluorescence time constant is a characteristic feature for each fluorophore, the fluorescence time constants are used to draw conclusions regarding the fluorophores excited in the object. While it is not possible based on prior art to distinguish different fluorophores, particularly of the ocular fundus, in a space-resolved two-dimensional representation due to the overlapping fluorescence spectra and the extremely low fluorescence intensity as a consequence of the limited radiant excitation power specified by safety regulations, the fluorophores can be distinguished according to their different fluorescence decay time. Surprisingly it was found that the extremely weak fluorescence intensities measurable from the ocular fundus represent very good conditions for time-correlated individual photon counting. The technique of time-correlated individual photon counting requires that by means of a laser excitation pulse, a fluorescence photon will be registered only with a probability of 0.1. The excitation pulse rate must be high so that an analyzable decay behavior can be registered within a correspondingly short time. The time regime for the time-correlated individual photon counting is controlled by the detected pulses of the fluorescence-exciting laser light and the fluorescence light created on the object. From the register contents of counters into which the detected fluorescence photon is read after each excitation pulse corresponding to the delay time between the excitation pulse and the detected fluorescence photon, the decay times of the transient autofluorescence caused by pulse excitation are determined for each site of the object, based on which the fluorophores are distinguished.
Further advantageous preferred embodiments are described in more detail hereinafter.